.alpha.-Amino acids play a central role in biology and are therefore of great importance in medicine. Isotopically-labeled amino acids and their derivatives have been increasingly used in biological research and in clinical testing over the last 50 years. Compounds which contain the radioactive isotope tritium (.sup.3 H=T) and the nonradioactive isotope deuterium (.sup.2 H=D) are among the most versatile and the least expensive probes available for monitoring processes which are important in biology, chemistry- and medicine. A great deal of effort has been expended to obtain specifically- deuteriated and -tritiated amino acids and numerous preparative procedures have appeared in the scientific literature. At present, large quantities of deuterium-labeled amino acids are used for nutritional testing, for labeling amino acid-derived metabolites of pharmaceuticals and agricultural chemicals, and for studies of protein structure. Conversely, compounds having less than the natural abundance level of deuterium (0.015%), by virtue of their low "background" interference, have utility in deuterium-sensitive analyses. Tritiated amino acids are widely used in biological, clinical and environmental analysis. For the purposes of this application, the term "amino acid" shall be understood to mean .alpha.-amino carboxylic acid.
Current methods for producing labeled amino acids fall into the categories of:
1) enzymatic/fermentation methodologies and PA0 2) chemical synthesis. PA0 a) alkylation of a nucleophilic glycine equivalent (e.g. anions derived from either acylaminomalonate or glycine ester imines) with a labeled alkylating agent, PA0 b) reductive amination with an ammonia equivalent and a labeled hydride reagent (e.g. NaCNBH.sub.2 T) PA0 c) exchange of an activated amino acid or acylamino acid with labeled water PA0 d) use of labeled molecular hydrogen for addition to a suitable multiple bond (usually carbon-carbon) or for replacement of a group (halogen, sulfur function, etc.) with the hydrogen label ("hydrogenolysis"). PA0 e) exchange of a ketoacid with labeled water, followed by conversion of the ketone carbonyl to an amino group ("reductive amination").
In general, enzymatic/fermentation methods (1) have the advantage of producing enantiomerically pure products. The drawbacks of these methods include the difficulty purifying products from complex incubation mixtures, and the limited number of possible amino acids which can be prepared by using known enzymes and microorganisms. Microbial fermentation in the presence of a labeled biological feedstock is the method of choice or generally-labeling the protein amino acids. "General" labeling implies the replacement of all of the atoms of a chosen element in a molecule with a specified isotope. Methods of type (1) are not well suited to site-specific labeling, that is, isotopic replacement of certain atoms of a given element, but not others in the same molecule. It is difficult to find conditions which can make the complex web of metabolic processes in living cells distinguish between different atoms of a given element in the same molecule, so as to specifically-label certain positions which are of particular interest.
Each amino acid contains a number of chemically-distinct hydrogen atoms. Substitution of all-, a select group-, or only one of the hydrogen atoms in a molecule will isotopic labels may be desirable, depending on the application. When designing an isotopic probe, choices as to which isotope to introduce and which atoms to substitute will depend on the problem to be solved. Chemical syntheses (2) can be used to introduce specific labels into any desired position in a molecule, but this may be difficult and/or expensive for certain products. Desirable features in a given isotopic synthesis may include the efficient use of readily available sources of label, a minimum number of chemical reactions and manipulations, simple purification procedures, the introduction of labels into specific- or multiple positions in the molecule as desired, and the ability to recover any unused isotopically-enriched reagents.
The .alpha.-amino acids comprise a large class of compounds, many, but not all of which, are naturally-occurring. A wide variety of functional groups are present in the side-chains of known amino acids, including hydrocarbon chains and rings, ionic functionalities (e.g. --COO-- and --NH.sub.3.sup.+), heterocyclic rings, and groups which are substituted with halogens, sulfur, phosphorus, selenium and many other elements. Because of this diversity, the amino acids vary a great deal in their physical and chemical properties. Differences in solubility are of particular concern when attempting to implement a general method of synthesis for these compounds, therefore any such method must be able to utilize a range of solvents.
Chemical reagents which are enriched in each of the heavy isotopes of hydrogen (.sup.2 H and .sup.3 H) are sold commercially for use as precursors in isotopic synthesis. The same group of reactions which are used to label amino acids with deuterium can also be used to introduce tritium, although the preferred methods will vary depending on the position to be labeled and the isotope to be used. Most such methods belong to one of four types:
Alkylation strategies (a) are preferred for introducing labels on sidechain groups (except for .beta.-hydrogen isotope introduction, see below), but they require a minimum of four synthetic steps using labeled materials and are therefore poorly suited for tritium labeling. Reductive amination with labeled hydride (b) requires a relatively costly hydride reagent, gives only modest yields (.ltoreq.50%), and only introduces one label where more may be desired. Amino acids and acylamino acids can be exchanged (c) in the .alpha.-position by using either carbonyl compounds or metal ion catalysts in either strongly acidic- or strongly basic media. Labeling in both the .alpha.- and .beta.-positions can be accomplished by using a combination of Al.sup.3+ and pyridoxal catalysts in isotopically-enriched HCl solution (Lemaster J. Labeled Compounds and Radiopharm., 1981, 18, 639), but for compounds with metal-coordinating groups on the sidechain (e.g. lysine) the exchange reaction can be slow and in all cases product must be purified from a complex reaction mixture. Molecular hydrogen (d) is an inexpensive source of label, but certain substrates for hydrogen addition are difficult to prepare (e.g. nonaromatic acylaminoacrylic acids), while preparation of other amino acid precursors, which contain hydrogenolyzable groups, is often a multistep process. The .beta.-hydrogens of .alpha.-ketoacids will exchange under base-catalysis, and a number of reductive amination methodologies are available for converting ketoacids to amino acids. The reductive amination method to be used is subject to one serious constraint: the reaction must not result in further exchange of the .beta.-hydrogens. In practice, this may exclude some commonly-used reductive amination procedures.